Author:

Samuel Gill

Mentor:

Chris Craney, Professor of Chemistry, Occidental College

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step in the pentose phosphate pathway. In addition, G6PD is functional only as a dimer and also provides the majority of a cell’s NADPH from NADP to be used in reductive processes. NADP is known to stabilize the dimer interface of G6PD. Different substrate analogues were added to G6PD to determine the occurrence of a hypothetical conformational change induced by these substrates. Using different substrate analogs to bring about conformational changes would allow a better understanding of the interactions between the subunits that grant the enzyme its activity. G6PD isolated from bakers yeast (Saccharomyces cerevisiae) served as a model system for experimentation with human G6PD. A number of limited proteolytic analysis were performed by saturating G6PD with different NADP analogues–NADP, thioNAD, and ADP– which were subsequently digested with subtilisin, a selective endoprotease. The resulting digestion was then analyzed via SDS-PAGE, producing a characteristic series of fragments for each digestion. In addition to NADP analogues, a digestion with a saturating amount of G6P was also performed to determine possible conformational changes when G6P is bound to G6PD. Comparisons of SDS-PAGE results indicate that the various NADP analogues tested do induce a conformational change in G6PD, evinced by a difference in fragmentation compared to a G6PD control digest. G6P, however, did not produce any difference in fragmentation, suggesting G6P does not affect G6PD dimer’s structure. Proteolysis are currently being performed on human G6PD to analogously investigate the dimer’s conformational changes with NADP analogues.